JP2003014685A - Biosensor and its manufacturing method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a biosensor in which a reaction layer containing an enzyme is rendered to a thin film in order to measure the concentration of a specific substrate in a biological component accurately in a short time. SOLUTION: The biosensor comprises an electric insulating substrate, an electrode system of a working electrode and a counter electrode formed on the substrate, and a reaction layer containing an enzyme formed on the electrode system or in the vicinity thereof. The reaction layer is formed by blowing a solution of a reagent composing the reaction layer onto the electrode system on the electric insulating substrate or in the vicinity thereof by electrospray method.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to quantification of substrates (specific components) contained in biological samples such as blood and urine, raw materials and products in the food industry, and samples such as fruit juice with high accuracy, quickly and easily. And a method for manufacturing the same.

[0002]

2. Description of the Related Art A biosensor has been proposed which can easily quantify a specific component (substrate) in a biological sample or food without diluting or stirring the sample solution. As one example thereof, in Japanese Patent Laid-Open No. 3-202764, an electrode system is formed on an insulating substrate by a method such as screen printing, and a reaction layer containing an oxidoreductase and an electron carrier is formed on the electrode system. Biosensors are disclosed.

This biosensor quantifies the substrate concentration in a sample as follows. First, by dropping the sample solution on the reaction layer of the biosensor, the reaction layer is dissolved,
An enzymatic reaction proceeds between the substrate in the sample solution and the redox enzyme in the reaction layer. With this enzymatic reaction, the electron carrier is reduced. After a certain time, voltage is applied to the electrodes of the sensor,
This reduced electron carrier is electrochemically oxidized, and the substrate concentration in the sample solution can be quantified from the oxidation current value obtained at this time. Regarding the amount of the enzyme contained in the reaction layer of the biosensor of this main constitution, the so-called dipping method is often used as the method for producing the reaction layer, and it is difficult to produce it as a thin film. In addition, it was difficult to prevent the produced layer from becoming non-uniform. Therefore, the amount of enzyme present in the reaction layer was excessive compared with the mass of the base contained in the sample to be quantified. Further, since the amount of the reagent contained in the reaction layer is large, there is a drawback that it takes time to dissolve the reagent layer.

[0004]

DISCLOSURE OF THE INVENTION The present invention solves the above problems, and provides a biosensor that more effectively utilizes an enzymatic reaction in a reaction layer by forming a uniform thin film. The purpose is to The present invention is
It is also an object to provide a method for manufacturing such a biosensor.

[0005]

In order to solve the above-mentioned problems, the biosensor of the present invention comprises an electrically insulating substrate, an electrode system having a working electrode and a counter electrode formed on the substrate,
And a reaction layer containing at least an enzyme formed on or near the electrode system, the reaction layer being a thin film formed by an electrospray method. The present invention also comprises an electrically insulating substrate, an electrode system having a working electrode and a counter electrode formed on the substrate, and a reaction layer containing at least an enzyme formed on or near the electrode system, There is provided a biosensor in which the uppermost layer of the reaction layer contains the enzyme and the uppermost layer is a thin film formed by an electrospray method.
The thickness of the reaction layer or the uppermost layer thereof is 10 nm to 1
It is preferably 000 nm. According to the present invention, a solution containing at least an enzyme is sprayed onto an electrically insulating substrate having an electrode system including a working electrode and a counter electrode on or near the electrode system by an electrospray method to form a thin reaction layer. The present invention relates to a method for manufacturing a biosensor, which has steps. The present invention provides a step of forming a layer containing a hydrophilic polymer or an electron carrier on or in the vicinity of the electrode system on an electrically insulating substrate having an electrode system including a working electrode and a counter electrode, and the above layer. A method for producing a biosensor, which further comprises the step of spraying a solution containing at least an enzyme thereon by an electrospray method to form a thin reaction layer.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION A biosensor according to the present invention is an electrically insulating substrate, an electrode system having a working electrode and a counter electrode formed on the substrate, and at least formed on or near the electrode system. A reaction layer containing an enzyme is provided. Then, the reaction layer is formed by spraying a solution of a reagent constituting the reaction layer on or near the electrode system on the electrically insulating substrate by an electrospray method. The reaction layer preferably contains an electron carrier. In another embodiment of the present invention, a part of the reagent forming the reaction layer, for example, an electron carrier, or a solution containing a hydrophilic polymer is spread on the electrode system or in the vicinity thereof by dropping or the like, After drying to form the first layer, a solution containing the enzyme is sprayed onto the first layer by an electrospray method and dried to form a second layer containing the enzyme. The first layer may be formed by an electrospray method.

As described above, the present invention forms the layer containing the enzyme as a thin film by the electrospray method. When the reaction layer is composed of a plurality of layers, the layer containing the enzyme is the uppermost layer. As a result, when the sample solution is supplied to the sensor, the enzyme is preferentially dissolved in the sample solution, and the enzymatic reaction proceeds rapidly.

Here, the electrospray method is an application of the electrospray ionization method used in mass spectrometry and the like, and a solution containing a compound to be contained in a reaction layer such as an enzyme or an electron carrier is sprayed using an electric field. Is the way. By forming the reaction layer using the electrospray method, each component constituting the reaction layer is made uniform,
Further, it becomes possible to apply it in the form of a thin film, and the solubility of the reaction layer of the sensor and the reactivity of the enzyme and the electron carrier can be improved.

The enzyme used in the present invention is
Examples thereof include glucose oxidase, glucose dehydrogenase, lactate oxidase, lactate dehydrogenase, fructose oxidase, cholesterol oxidase, cholesterol dehydrogenase, cholesterol esterase, mutarotase, inverdase, ascorbate oxidase, alcohol oxidase and the like. It is also possible to use one kind or a mixture of two or more kinds of these enzymes. The reaction layer of the biosensor of the present invention may include an electron carrier as described above. In this case, examples of the electron carrier include ferricyanide salt, p-benzoquinone and its derivative, phenazine methosulfate, methylene blue, ferrocene and its derivative. The electron carrier is one or two of these.
More than one seed is used. When the above-mentioned electron carrier is not contained in the reaction layer, it is possible to quantify the base mass by utilizing oxygen or oxygen peroxide in the solution.

Solvents for dissolving enzymes and electron carriers in the solution using the electrospray method are water, phosphate buffer, Tris-hydrochloride buffer, phthalate buffer, acetate buffer, various aqueous solutions such as sodium chloride solution, Alternatively, it is preferably a single solution or a mixed solution of two or more selected from organic solvents such as methanol, ethanol, toluene, and acetone. The reaction layer of the biosensor of the present invention may contain a hydrophilic polymer in addition to the above-mentioned enzymes and electron carriers. By adding a hydrophilic polymer to the reaction layer, peeling of the reaction layer from the surface of the substrate or the electrode system can be prevented. The hydrophilic polymer also has an effect of preventing cracks on the surface of the reaction layer, and is effective in increasing the reliability of the biosensor. Further, the electrode system may be coated with a hydrophilic polymer, and the same effect as above can be obtained.

Such hydrophilic polymers include carboxymethyl cellulose, hydroxyethyl cellulose,
Hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, ethyl hydroxyethyl cellulose,
Examples thereof include carboxymethyl ethyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids such as polylysine, polystyrene sulfonic acid, gelatin and its derivatives, acrylic acid or its derivatives, polymers of maleic anhydride or its salts, starch and its derivatives, and the like. Particularly, carboxymethyl cellulose is preferable. As a method for measuring the oxidation current, there are a two-electrode method in which only a measurement electrode and a counter electrode are used, and a three-electrode method in which a reference electrode is added. The three-electrode method enables more accurate measurement.

[0012]

EXAMPLES The present invention will be described in detail below with reference to specific examples. FIG. 1 is an exploded perspective view of the biosensor according to the present invention with the reaction layer removed. Reference numeral 1 denotes an insulating substrate made of polyethylene terephthalate. The leads 2 and 3 are formed on the substrate 1 by printing a silver paste by screen printing. An electrode system including a working electrode 4 and a counter electrode 5 made of a conductive carbon paste containing a resin binder, and an electric insulating layer 6 made of an electrically insulating paste are further formed on the substrate 1 by the same printing method. is there. The electrically insulating layer 6 makes the areas of the exposed portions of the working electrode 4 and the counter electrode 5 constant and partially covers the leads. As the material of the lead and the electrode, platinum, gold, palladium and the like are used in addition to silver and carbon. FIG. 2 is a schematic vertical sectional view of the above biosensor.

A reaction layer 7 containing an enzyme and an electron carrier is formed on an insulating substrate 1 having an electrode system formed as shown in FIG. A biosensor is assembled by adhering a cover 9 and a spacer 8 sandwiched between the substrate 1 and the cover 9 to the insulating substrate 1 in a positional relationship as shown by the alternate long and short dash line in FIG. The spacer 8 is formed with a slit 10 forming a sample liquid supply path, and the cover 9 is formed with an air hole 11. When the substrate 1, the cover 9 and the spacer 8 are combined together, a space portion serving as a sample supply path is formed between the substrate 1 and the cover 9. In this space, the open end 12 of the slit 10 serves as a sample supply port, and the terminal end serves as an air hole 11.

Although the cover member is formed by the combination of the cover 9 and the spacer 8 here, it may be formed by a single member provided with a groove corresponding to the slit 10 of the spacer 8. Further, the reaction layer does not necessarily have to be formed on the electrode system, as long as it is formed on a portion exposed in the sample supply path, and the sample solution may dissolve the reaction layer and reach the electrode system. Good.

Example 1 On the electrode system of the substrate 1 of FIG.
Potassium ferricyanide and glucose oxidase (E
An aqueous solution containing C1.1.3.4: hereinafter abbreviated as GOD) was sprayed by an electrospray method and dried to form a reaction layer 7. This electrospray method was carried out as follows. The substrate 1 is set on a metal plate electrode larger than this, a nozzle for ejecting the solution is set above the electrode system of the substrate 1, and the needle electrode in the nozzle is made negative to form a metal plate electrode. A voltage of 3 kV was applied between them, and the aqueous solution was sprayed on the electrode system of the substrate 1 from a nozzle for 10 seconds, and then dried. The amount of potassium ferricyanide contained in the reaction layer 7 thus formed is 0.1 mg per 1 cm 2, and G
The amount of OD was 5 μg. The thickness of the formed thin film reaction layer was 100 nm.

Next, a sensor was assembled with the positional relationship shown in FIG. 2 and the characteristics of the glucose sensor were evaluated. When the sample solution containing glucose is supplied to the reaction layer 7, glucose in the sample solution is oxidized by GOD. At the same time, the electron carrier in the reaction layer is reduced. Subsequently, 55 seconds after supplying the sample liquid, a voltage of +0.5 V was applied to the working electrode 4 with reference to the counter electrode 5 to oxidize the reduced form of the electron carrier. Then, the current value after 5 seconds was measured. The measurement time in this case, that is, the time from the supply of the sample to the measurement of the oxidation current value is set to 1 minute. This current value is proportional to the concentration of the produced electron carrier, that is, the substrate concentration in the sample solution, so that the glucose concentration of the sample solution can be determined by measuring this current value. 0, 180, 360, and 540 mg /
Prepare a sample of each glucose concentration of dl and set the measurement time to 3
As a result of measuring the response current value of the sensor with respect to 0 second, there was a certain correlation between the response current value and the glucose concentration, and good linearity was shown. Also,
Even when the measurement time was 10 seconds, the relationship between the response current value and the glucose concentration showed good linearity.

Example 2 An aqueous solution containing potassium ferricyanide was prepared, which was dropped on an electrode and dried to form a first layer on the electrode system. The amount of potassium ferricyanide contained in this first layer was 1 mg per square centimeter. Next, an aqueous GOD solution was prepared, and this aqueous solution was sprayed on the first layer by the same electrospray method as in Example 1 and dried to form the second layer. However, the applied voltage was 2.5 kV and the spraying time was 2.5 seconds. GOD contained in this second layer
Was 1 μg per 1 cm 2 of the reaction layer. The thickness of the second layer of the produced thin film was 20 nm. As a result of measuring the sensor response current value to the glucose standard solution by the same method as in Example 1, the response current value showed good linearity with respect to the glucose concentration at both the measurement times of 30 seconds and 10 seconds.

Comparative Example 1 Potassium ferricyanide and G
An aqueous solution containing OD was dropped on the electrode and dried to form a reaction layer. The amount of potassium ferricyanide contained in this reaction layer was 1 mg per square centimeter,
The amount of GOD was 50 μg. The thickness of the produced reaction layer was 20 μm. As a result of measuring the sensor response current value for the glucose standard solution by the same method as in Example 1, when the measurement time was 30 seconds, the relationship between the response current value and the glucose concentration showed good linearity. But,
When the measurement time was 10 seconds, the relationship between the response current value and the glucose concentration did not show linearity in the high concentration range.

Example 3 Similar to Example 2, a first layer containing potassium ferricyanide was formed. Next, an aqueous solution containing cholesterol esterase and cholesterol oxidase was prepared, and the solution was sprayed on the first layer by an electrospray method and then dried to form a second layer. The applied voltage in this case is 2k
V and the spraying time were 20 seconds. The amounts of enzymes contained in the second layer 7b were 20 μg of cholesterol oxidase and 50 μg of cholesterol esterase per 1 cm 2 of the reaction layer. The thickness of the second layer thin film was 150 nm.

A cholesterol sensor was produced in the same manner as the glucose sensor, and the sensor response current value for the sample solution containing cholesterol was measured. As a result, the response current value at the measurement time of 3 minutes showed good linearity with respect to the cholesterol concentration. showed that. In addition, good linearity was shown with respect to the cholesterol concentration even at the measurement time of 1 minute.

Comparative Example 2 On the electrode system of the substrate 1 of FIG.
Drop a mixed aqueous solution of potassium ferricyanide, cholesterol oxidase, and cholesterol esterase,
The reaction layer 7 was formed by drying. The amount of potassium ferricyanide contained in this layer 7 was 1 mg per 1 cm 2 of the reaction layer, the amount of cholesterol oxidase was 0.1 mg, and the amount of cholesterol esterase was 0.5 mg. The thickness of the reaction layer was 50 μm. Next, the response current value of the sensor was measured by the same method as in Example 3. As a result, at the measurement time of 3 minutes, there was a certain correlation between the response current value and the cholesterol concentration, and good linearity was shown. This response linearity was almost the same as that shown in Example 1. However, the response linearity was not shown at the measurement time of 1 minute.

Example 4 On an electrode system made of platinum,
An aqueous solution containing glucose oxidase (GOD) was sprayed by an electrospray method and dried to form a reaction layer 7. The amount of GOD contained in the reaction layer 7 formed by spraying at an applied voltage of 2 kV for 5 seconds was 3 μg.
The thickness of the formed thin film reaction layer was 50 nm.
When a sample solution containing glucose is supplied onto this electrode system,
Glucose in the sample solution is oxidized by GOD to form hydrogen peroxide. Therefore, 10 seconds after the sample liquid was supplied, a voltage of +1.0 V was applied to the working electrode 4 with the counter electrode 5 as a reference, and the current value after 5 seconds was measured. Since this current value is proportional to the substrate concentration in the sample solution, the glucose concentration in the sample solution can be determined by measuring this current value. 0, 180, 360, and 540 mg / dl
As a result of preparing a sample of each glucose concentration of, and measuring the response current value of the sensor for each when the measurement time was 30 seconds, there was a certain correlation between the response current value and the glucose concentration, which was good. It showed excellent linearity. Even when the measurement time was 10 seconds, the relationship between the response current value and the glucose concentration showed good linearity.

The reaction layer forming method using the electrospray method can be made thinner than the forming method by the dip method. Therefore, the dissolution of the reagent containing the enzyme contained in the reaction layer becomes smooth. As a result, the measurement time can be shortened. If the thickness of the thin film is 10 nm or less, it is difficult to produce a uniform film. Even if it can be manufactured, it does not function sufficiently as a sensor because the amount of the reagent contained therein is insufficient. On the other hand, the thin film of the reaction layer has a thickness of 100.
When it is 0 nm or more, the solubility of the reaction layer is lowered and the measurement in a short time becomes difficult. Therefore, the thickness of the thin film should be 10
It is effective to set the thickness from nm to 1000 nm.

Although the glucose sensor and the cholesterol sensor have been described in the above embodiments, the glucose sensor or the cholesterol sensor using other enzymes, the lactate sensor, the fructose sensor, the sucrose sensor,
It goes without saying that the present invention can be applied to each sensor such as an alcohol sensor and an ascorbic acid sensor.

[0025]

As described above, according to the present invention, blood,
It is possible to obtain a biosensor capable of quantifying a substrate contained in a biological sample such as urine or a sample such as a raw material or a product in the food industry with high accuracy, quickly and easily.

[Brief description of drawings]

FIG. 1 is an exploded perspective view of a biosensor according to an embodiment of the present invention, excluding a reaction layer.

FIG. 2 is a schematic vertical sectional view of the biosensor.

[Explanation of symbols]

1 Electrically insulating substrate A few leads 4 Working pole 5 opposite poles 6 insulating layers 7 Reaction layer 8 spacers 9 cover 10 slits 11 air holes

Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/483 G01N 27/30 353B 353P 27/46 336H 336G 338 (72) Inventor Miwa Hasegawa Kadoma City, Osaka Prefecture 1006 Address Matsushita Electric Industrial Co., Ltd. (72) Inventor Kiichi Watanabe 1006 Kadoma, Kadoma City, Osaka Prefecture Matsushita Electric Industrial Co., Ltd. (72) Inventor, Toshihiko Yoshioka 1006 Kadoma, Kadoma City, Osaka Matsushita Electric Industrial Co., Ltd. F term (reference) 2G045 AA13 AA16 CA25 CA26 DA31 DA69 FB01 FB05 GC20 4B029 AA07 AA21 BB16 CC03 CC11 FA12 4B033 NA03 NA23 NA24 NB34 NB45 NB63 NB66 NC04 NC06 NC06 NC12 NC16 ND05 ND16 4B06QQQQQAQQQQAQ

Claims (7)

[Claims] 1. An electrically insulating substrate, an electrode system having a working electrode and a counter electrode formed on the substrate, and a reaction layer containing at least an enzyme formed on or near the electrode system. A biosensor, wherein the reaction layer comprises a thin film formed by an electrospray method. 2. An electrically insulating substrate, an electrode system having a working electrode and a counter electrode formed on the substrate, and a reaction layer containing at least an enzyme formed on or near the electrode system, A biosensor, wherein the uppermost layer of the reaction layer contains the enzyme and the uppermost layer is a thin film formed by an electrospray method. 3. The reaction layer contains an electron carrier.
Alternatively, the biosensor according to item 2. 4. The thickness of the reaction layer is 10 nm to 10 nm.
The biosensor according to claim 1, which has a thickness of 00 nm. 5. The thickness of the uppermost layer is 10 nm to 10 nm.
The biosensor according to claim 2, which has a thickness of 00 nm. 6. On or near the electrode system on an electrically insulating substrate having an electrode system including a working electrode and a counter electrode,
A method for producing a biosensor, comprising a step of forming a thin film reaction layer by spraying a solution containing at least an enzyme by an electrospray method. 7. On or near the electrode system on an electrically insulating substrate having an electrode system including a working electrode and a counter electrode,
And a step of forming a layer containing a hydrophilic polymer or an electron carrier, and a step of spraying a solution containing at least an enzyme onto the layer by an electrospray method to form a thin reaction layer. And a method for manufacturing a biosensor.